The development of an effective AIDS vaccine remains one of the highest priorities in HIV research. Studies of HIV pathogenesis, vaccine design and antiretroviral treatments require an appropriate animal model. However, the existing SIV/macaque model has major limitations since (i) prototypic SIVmac strains are too pathogenic, (ii) do not represent mucosally transmitted viruses, and (iii) have been extensively passaged in vitro and in vivo. We have recently identified a large set of new SIVsmm strains which mirror HIV-1 group M viruses in their genetic diversity. Moreover, preliminary in vivo results show that infection of RMs with these SIVsmm strains more closely reproduces the natural history of HIV-1 in humans. Within this HIVRAD consortium, we thus propose to use these new SIVsmm strains to generate new (molecularly cloned) challenge viruses for AIDS vaccine and pathogenesis studies. Project 1 will use a dose-escalation strategy to infect 21 Indian rhesus macaques (RMs) with genetically diverse SIVsmm strains using intravenous (iv), intrarectal (ir) and intravaginal (ivag) routes. Project 2 will then employ single genome amplification (SGA) techniques to infer, and subsequently clone, transmitted/founder (T/F) virus(es) from all of these animals. Following detailed in vitro characterization, a subset of clones will be selected for in vivo competition (n=18) and pathogenesis (n=8) studies which will be performed by Project 1. Specific Aims include: 1. To generate high titer plasma stocks of genetically divergent SIVsmm strains without in vitro adaptation for subsequent mucosal and intravenous transmission studies. 2. To infect RMs by intravenous, intrarectal and intravaginal routes with physiologically relevant doses of genetically divergent SIVsmm strains to allow for the identification of transmitted founder (T/F) viruses: 3.; To identify SIVsmm clones with, preferential mucosal transmissibility and replication fitness by conducting an in vivo competition experiment. 4. To characterize the selected SIVsmm clones for in vivo replication kinetics, pathogenicity and suitability as vaccine challenge stocks. We expect these studies to generate new infectious molecular clones of SIVs with biological properties that more faithfully recapitulate the transmission, pathogenic and diversity of HIV-1 in humans.